Project Summary Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that affects 2.3 million people globally. MS was traditionally thought to be a T cell mediated disease since autoreactive T cells, a kind of immune cell, against myelin has been found and their role carefully studied in mouse models of MS. However, B cell depleting therapies have had excellent clinical success in treating MS, suggesting a key role for B cells, another kind of immune cell, in MS disease. However, it is unclear how these therapies affect other key immune cells in the blood or in the cerebrospinal fluid (CSF), the fluid that bathes the CNS. Characterization of these changes is crucial to understanding how these therapies work and determining if more targeted approaches can be developed. Our goal is to characterize the full repertoire of cells in the CSF and blood of MS patients, and determine how these populations are altered longitudinally by treatment with Ocrelizumab. By leveraging a unique therapeutic opportunity, we will identify the spatiotemporal shifts that occur in response to decreased frequencies B cells. To accomplish our goal, we will: 1) Define the composition of CSF as compared to blood from healthy and MS donors, 2) track single cell alterations in blood and CSF of MS patients over the course of Ocrelizumab treatment, and 3) develop and implement unique computational analyses to identify cell states and genes that correlate with disease, treatment, and anatomical compartment. We propose to implement a new method for analyzing single cells, called Seq-Well, which will allow us to transcriptionally profile thousands of single cells in parallel. This method is well suited for profiling rare and low input clinical samples like those from CSF. We hypothesize that alterations in the total pool of B cells will lead to global changes in the immune cell landscape, and specifically, will impact the frequency and transcriptional state of T cells. This work is fundamental to human health because it will shed light on the mechanism of B cell depleting therapies, should enable the development of immune monitoring strategies, and will refine models of CNS-peripheral cross talk. Specifically, our work will also identify key gene modules underlying clinically relevant axes, potentially suggesting better biomarkers and treatment paradigms while improving our understanding of immune system dysfunction in MS. Finally, the unique data sets and analyses that will be generated as part of this work will contribute broadly to our understanding the CSF and result in new analytical pipelines that integrate various forms of single-cell data.